عنوان مقاله [English]
Introduction: Pyrus communis L. cv. Natanz is a popular pear cultivar in Iran because of its customer-friendly attribute due to its excellent characteristics. Pear own-rooted plants has better traits such as high vigorous in growth, low levels on tree losses and damaging by insects rather than grafted plants. Meristem culture widely used for micropropagation, in vitro germplasm preservation, and virus eradication purposes in pear. As pear is belonged to difficult-to-root fruit tree cultivars perhaps the rooting stage is the most important stage in propagation process, yet most difficult phase during the in vitro propagation procedure. In vitro rooting of micro-cuts was varied by genotypes (cultivars), type and concentration of auxin, the method of root induction and formation, different additional materials such as PVP, polyamines, and so on. This study was aimed to investigate the effect of different levels of BAP and Fe-EDDHA on shoot proliferation, BAP and GA3 on meristem establishment, and IBA and NAA on micro-cut rooting of pear cv. Natanz in in vitro condition.
Materials and Methods: Vegetative buds were taken from current growth shoots of Pyrus communis cv. Natanz from Pear collection orchard (25.36 E, 58.54 N, and ASL altitude 1380 m) of Agricultural and Natural Resources Research and Education Centre of Semnan Province (Shahrood city). In the first experiment, new shoots of active buds after 4 weeks grown in PMI medium (MS ×1.5 CaCl2. 2H2O, KH2PO4 and MgSO4. 7H2O) + 1 mg.l-1 BAP were transferred to PMI medium containing different levels of BAP (0.5, 1, 1.5 mg.l-1) and Fe-EDDHA (0, 100, 150 and 200 mg.l-1). In the second experiment, meristems (containing two newest leaf primordia) was excited from in vitro shoots and incubation on MS media containing BAP (0.5, 1, and 1.5 mg.l-1) and GA3 (0.1 and 0.5 mg.l-1) + 0.1 mg.l-1 IBA. Meristems were kept in dark for 4 days then were transferred to growth chamber with photoperiod 16/8 hrs. light/dark. Different concentrations and combinations of two auxins were used for root induction of micro-cuts in third experiment. 1000, 2000, 3000, and 4000 mg.l-1 of IBA or NAA and two combination solutions of them (1000 IBA+1000 NAA, and 2000 IBA+2000 NAA, mg.l-1). Shoots were immersing dip in solutions for 5 seconds then transfer to PGRs-free PMI medium and kept them to growth chamber. Data of all experiments were analyzed according by completely randomized design (CRD) with five replications. BAP (3 levels) and Fe-EDDHA (4 levels) for experiment 1; BAP (3 levels) and GA3 (2 levels) for experiment 2 were considered as factorial. SAS (v. 9.1) was used for analysis and means were compared with LSD test at 5% of probability level.
Results and Discussion: Proliferated shoot number was affected by BAP (p≤0.01) and Fe-EDDHA (p≤0.05) concentrations and also interaction of them (p≤0.05), while BAP (p≤0.01) was caused elongation of proliferated shoots and Fe-EDDHA had no effect. BAP (p≤0.05), Fe-EDDHA (p≤0.01) concentrations and BAP×Fe-EDDHA (p≤0.01) interaction had significant effect on leaf production. Shoot tip necrosis was shown in shoots grown in all media based on BAP concentration with different intensities (p≤0.05). Vegetative growth was counted as a power index of medium that in our experiment was under influence of BAP concentrations (p≤0.01), Fe-EDDHA (p≤0.05) and BAP×Fe-EDDHA interaction (p≤0.05). Shoots were proliferated (5.50 shoot.explant-1) and elongated in PMI medium containing 1.5 mg.l-1 BAP with no Fe-NaEDDHA while the lower concentrations of both BAP and Fe-NaEDDHA caused the higher mature leaf production. PMI media containing 1 mg.l-1 BAP + 150 mg.l-1 Fe-NaEDDHA is recommended for Natanz shoot proliferation because of the highest vegetative growth and highest quality in proliferated shoots. MS medium with 0.5 mg.l-1 BAP+ 0.5 mg.l-1 GA3 (81%) and 1 mg.l-1 BAP + 0.1 mg.l-1 GA3 (63%) had the highest meristem establishment, respectively. The established meristems naturally grown in medium supplement with 0.5 mg.l-1 BAP + 0.5 mg.l-1 GA3+0.1 mg.l-1 IBA. Different types of auxin and their concentrations had significantly effect on Natanz pear cultivar micro-cut rooting (p≤0.05). NAA induced rooting in lower concentrations while IBA had positive effect on rooting with concentration increasing. Micro-cuts were rooted via quick dip in 1000+1000 mg.l-1 (IBA+NAA) solution followed by incubation in PMI medium. The rooted shoots well adapted to environmental condition.
Conclusion: Important steps of in vitro propagation of pear is optimized in this experiment. MS medium containing 0.5 mg.l-1 BAP+0.5 mg.l-1 GA3+0.1 mg.l-1 IBA had suitable for meristem establishment. To produce in vitro healthy proliferated shoots of pear cv. Natanz using PMI medium supplement with 1 mg.l-1 BAP+150 mg.l-1 Fe-NaEDDHA is recommended. Micro-cuts were rooted easily by quick immersion of the end of micro-cuts in 1000+1000 mg.l-1 (IBA+NAA) solution for 5 seconds then incubation in PGRs-free medium.